Cell cultures are used in the biotechnology industry principally to produce recombinant proteins and monoclonal antibodies. Recently however, a technology has been developed to culture different types of core cells for use in cell therapy, in combination or not with gene therapy.
To successfully culture and maintain the different cell lines, a medium is required that imitates the conditions of the internal medium in which these cells are found in vivo. Generally, the culture medium consists of a basal medium which provides the pH, nutrients and salts and to which a series of supplements may be added to ensure cell proliferation. One of the most widely used supplements is animal blood serum. As a result of the animal origin of the serum there may be serious restrictions for its use to obtain products for use in humans, because residues of animal origin are recognised as foreign antigens and may cause adverse reactions in the recipients. Furthermore, animal sera have the drawback of lacking a defined composition, as there is a great variability between manufacturers because different animal breeds are used. In addition, because of the method by which they are obtained, there is great variability between batches, which means that wide variations may occur in growth capacity and in the production of a particular culture.
The main difficulty in establishing cell lines is obtaining suitable nutritive media which are capable of replacing the natural medium, such as embryo extracts, protein hydrolysates or sera. A first group of media, such as the Eagle basal medium (MEM) (Eagle, H. (1955) “The specific aminoacid requirements of mammalian cells (strain L) in tissue culture”. J. Biol. Chem. 214: 839) and the more complex 199 medium of Morgan et al. (Morgan, J. G., Morton, J. H. and Parker, R. C. (1950) “Nutrition of animal cells in tissue culture. I Initial studies on a synthetic medium”. Proc. Soc. Exp. Biol. Med. 73: 1) were defined media but they require a serum supplement of between 5% and 20%.
To eliminate the contribution of undefined complex media more complex media have been formulated such as NCTC 109 (Evans, V. J., Bryant, J. C., Fioramonti, M. C., McQuilkin, W. T., Sanford, K. K., and Earle, W. R. (1956) “Studies of nutrient media for tissue C cells in vitro. I A protein-free chemically defined medium for cultivation of strain L cells” Cancer Res. 16: 77), 135 (Evans, V. J. and Briant, J. C. (1965) “Advances in tissue culture at the National Cancer Institute in the United States of America” in “Tissue culture”, edited by C. V. Ramakrishnan, W. Junk, The Hague, pp. 145-167), Ham F10 and F12 (Ham, R. G. (1963). “An improved nutrient solution for diploid Chinese hamster and human cell lines”. Exp. Cell Res. 29: 515, Ham, R. G. (1965) “Clonal growth of mammalian cells in a chemically defined synthetic medium” Proc. Natl. Sci. USA 53: 288), MCDB series (Ham, R. G. and McKeehan, W. L. (1978) “Development of improved media and culture conditions for clonal growth of normal diploid cells” In vitro 14: 11-22) and hormone-supplemented Sato media (Barnes, D. and Sato, G. (1980) “Methods for growth of cultured cells in serum-free medium” Anal. Biochem. 102: 255-270).
The recommended approach in order to establish a defined medium is to begin with a rich medium, for example Ham F12, supplemented with a high concentration of serum (20%) and test supplements for reducing the quantity of serum until it can be reduced or eliminated.
After years of investigation into the composition of media, they are still selected empirically.
The principal media used and their applications are:                Eagle Basal Medium (EBM). This is a basic medium with only essential aminoacids. It always requires supplementation with 10% foetal calf serum. It is used for growing mouse fibroblasts and HeLa cells.        Eagle's Minimum Essential Medium (MEM). This is the most commonly used medium, which contains more aminoacids in a higher concentration than EBM. It is used for almost any type of culture and requires the addition of serum (10%).        R.P.M.I. 1640. This is a medium designed for growing lymphoblasts and leukaemia cell lines in suspension. With suitable supplements, it has a wide range of applications.        Dulbecco Modified Eagle Medium (DMEM). It contains four times the concentration of aminoacids and vitamins as EBM. It is used for selecting hybridomas supplemented with HAT (hypoxanthine-aminopterin-thymidine) or HT (hypoxanthine-thymidine).        Iscove modified DMEM (IMDM). This is a very complete medium with a formulation that includes bovine albumin, transferrin and selenite, among other elements. It is very useful in the culture of lymphocytes in serum-free medium. It is also used for other cell types, but in this case requires low concentrations of serum.        McCoy 5a medium. Designed for growing diploid cell lines of both rats and humans.        Leibovitz L-15 medium. Used for the culture of viruses.        Ham F-10 medium. It is used for growing human cell lines and must be supplemented with proteins and hormones. It contains metals such as Fe, Cu and Zn. It is used for amniotic cell culture.        Ham F-12 medium. It is used for growing cell lines with protein supplements. Combined with IMDM it can be used as a serum-free medium.        Medium 199. It is used very widely for undifferentiated cell culture and for studying chromosome disorders.        
All culture media consist of the following elements:
1. Balanced saline solutions (BSS).
2. Aminoacids.
3. Vitamins.
4. Other organic supplements with a low molecular weight (nucleosides, Krebs cycle intermediates, pyruvate, lipids).
5. Hormones and growth factors (serum).
6. Contaminant growth inhibitors (antibiotics and antifungals).
In undefined media, the serum usually provides hormones and growth factors. The types of serum used are calf serum (CF), foetal calf serum (FCS), horse serum (HS) and human serum (HuS). The most widely used is calf serum, while foetal calf serum is used in more demanding lines and human serum is used in human lines.
The use of serum is problematic as despite the composition of the serum being known in part, there are a great many components present in variable quantities which may significantly influence the culture. In addition, the serum varies from batch to batch, due to the variability of the technique for obtaining blood, the method of obtaining the serum (blood coagulation) and the conditions for separating the serum, as well as the difference between the various sources of serum. Moreover, each change of serum batch requires a series of tedious and costly checks. Further, if the products of the culture medium have to be purified, the presence of variable components in the serum makes these processes significantly more difficult.
Some serum factors, such as the platelet-derived growth factor (PDGF), stimulate the proliferation of fibroblasts, which may be a problem in establishing specialised primary cultures, particularly if there is a wide variation in their content.
On the whole, the inclusion of serum in culture media is a significant drawback to standardising experimental protocols and cell production. Great efforts are therefore being made to establish media with a defined composition for cell growth. As well as the reproducibility sought, this allows selective media to be established in which the cell type required can grow. The disadvantage of these media is that in many cases cell growth is lower and cell lines remain viable for fewer generations.
Various attempts have been made to produce supplements for cell culture media which avoid the problems posed by the sera used at present. Among these, various fractions from human plasma have been tested and there are significant contradictions in the prior art as to the usefulness of the different human plasma fractions.
In general, different human plasma fractions originating from plasma fractionation using the Cohn method have been tested (Cohn E. J. et al; J Am Chem Soc, 1946) and variations thereof (Kistler and Friedli, Nischmann; in Curling, J M ed, Methods of plasma fractionation, Academic Press 1980), all these tests being directed towards the use of the different precipitates obtained in the fractionation, particularly the II+III, III, IV (IV1 or IV4) and V fractions, or their equivalents in different variants of the fractionation method.
The initial aim in separating the above-mentioned fractions (precipitate) is to obtain a precipitate enriched with a particular protein as a starting point for the purification of said protein, for example γ-globulins and α and β-globulins in the case of the II+III or III fractions; α-globulins and transferrin in the IV fraction and albumin in the V fraction. These fractions therefore usually have a single type of protein, other proteins being present as impurities, usually in far smaller quantities. The use of these fractions in cell cultures involves adding to the culture medium a major protein type and a variety of proteins which accompany it as impurities, among which must be present those required by the cells in the culture medium if the use of said material is to be successful. Until now this has been an obvious but inefficient way of supplementing cell culture media. This is certainly the cause of the disparity in the results obtained, as small variations in the fractionation method will not significantly affect the recovery of the major protein, but may introduce great variability in the recovery of the various accompanying proteins. For example, the II+III fraction using the Cohn method is obtained at an ethanol concentration of between 20% and 25% at −5° C. and a pH of 6.9. Using the Nitschmann method, starting with equivalent material, it is precipitated at an ethanol concentration of 19% at −5° C. and a pH of 5.8. With this variation in the pH, fractions are obtained with different characteristics, particularly in the content of γ-globulins and other accompanying proteins.
Furthermore, the use of supernatants, compared with the use of precipitated fractions, has additional advantages, including that of maintaining a high albumin concentration in the medium and, above all, avoiding the loss of components (for example hormones, cytokines, lipids, etc.) which are important for cell growth and which may not be present in the precipitates or may lose their functionality (be inactivated) in the presence of alcohol concentrations of more than 20%, such as those used to precipitate the II+III fraction in certain conditions (25%) or the IV1+IV4 and V fractions (40% ethanol).
Document EP0143648 (Macleod) describes the use of the II+III, III, IV1 and IV4 fractions as supplements for culture media instead of animal serum. It also states that neither the II fraction nor the V fraction is useful for this application. In all cases, this document refers exclusively to fractions precipitated in the Cohn fractionation or its variants. In addition, the document discloses a method for obtaining material suitable for supplementing culture media from the above-mentioned fractions. This method consists basically of suspending the precipitate in water and homogenising said precipitate. Next, the pH is adjusted to achieve better protein dissolution and produce the physiological conditions for cell growth. Further, this method also considers the elimination of the γ-globulin present by precipitation with polyethylene glycol and the separation of material of low molecular weight by molecular-exclusion chromatography. The effectiveness of the material prepared in this way is therefore very dependent on the specific preparation method.
Another document by Macleod (Advances in Biochemical Engineering/Biotechnology, Vol. 37; 1988) indicates that the II, III and IV fractions have little or no effect on cell growth, possibly due to the presence of growth inhibitors and focuses attention on the IV4 fraction as the ideal material to supplement culture media.
EP 0 440 509 (Macleod) describes a supplement for cell culture media based on the Cohn's IV fraction or II+III fraction, and the process for obtaining it. Using the process described, a product is obtained which is substantially free from immunoglobulins, is virus-inactivated and stable. The immunoglobulin is separated by precipitation with polyethylene glycol and viruses are inactivated by pasteurization, in the presence of sorbitol as a stabiliser. This process is particularly applicable to the IV (IV1 or IV4) fraction.
Document EP 0264748 (Antoniades) describes the supernatant of the Cohn V fraction as a supplement for cell culture media instead of animal serum. According to this document, the advantage of this material is that there is no cost and it can be heated to 60° C. for 20 hours.
The supernatant of the V fraction is the final waste material from the Cohn fractionation and therefore its use clearly has no economic cost to detract from the benefits of said fractionation. This material has a high ethanol content (40%), which is highly toxic and which this document does not state must be eliminated. Furthermore, for the treatment described at 60° C. the protein present would have to be stabilised, but this detail is also omitted from the document, making the invention difficult to produce as described. Nor should the denaturing effect of ethanol at so high a concentration as in this case be underestimated.
WO 94/18310 (Mankarious) describes a method for producing a supplement for cell culture media from the Cohn IV4 fraction. This method looks at the suspension and subsequent clarification of the IV4 fraction and also the inactivation or elimination of viruses by pasteurization and/or filtration.
Patent GB 2 166 756 A describes a method for the culture of spleen cells for the production of interferon. This method is based on an RPMI culture medium which is supplemented by a serum fraction obtained from serum or plasma from which the fibrinogen has been eliminated and which is characterised in that the prealbumin and gammaglobulins have been removed (page 1, lines 30-34). In a preferred embodiment, this fraction is purified by precipitation with alcohol between 10% and 20%, at a pH of 5.85 (page 1, lines 37-38). In a particular embodiment (obtaining the EPF fraction), the supernatant used as a culture medium is obtained at an ethanol concentration of 19% and a pH of 5.85 (page 2, lines 44-45). The supernatant obtained is dialysed in order to eliminate the ethanol, and is subsequently lyophilised to preserve it. Optionally, it is lyophilised with no prior dialysis, and consequently the ethanol is also eliminated. According to the inventors, by using a culture medium (RPMI) supplemented with said fraction, excellent interferon production is obtained. In the alcohol precipitation of the plasma, the process conditions (ethanol concentration, pH, ion strength of the medium, temperature and protein concentration) must be carefully set to maintain particular proteins in solution and insolubilize others so that they are precipitated and can thus be separated. Variations in one or more of said conditions will produce significant differences in the product obtained, the ethanol concentration and pH being the factors determining the composition of the precipitate and supernatant obtained.
Other attempts have been made to supplement cell culture media using serum or serum fractions of human origin, instead of foetal calf serum, as shown in documents WO 2004/055174, EP 1820852 or CA 1177424. The serum obtained from complete blood or from plasma cannot be compared with a specifically defined and characterised plasma fraction and has the drawback of lacking reproducibility between batches. In addition, none of these documents succeeds in defining and characterising the exact composition of the material used. Therefore the majority of the problems associated with the use of serum of animal origin continue to arise.
Kwok et al. disclosed the use of plasma from pregnant women instead of foetal calf serum for the culture of specific cell types, attributing its effect to the presence of unknown factors. He also postulates a synergic effect of human albumin in this type of culture. It is clear to a person skilled in the art that by using plasma from pregnant women the presence is sought of cell growth factors comparable to those of foetal serum, but the homogeneity of this material is not comparable to plasma mixtures from thousands of donors, as described below.
As described hereabove, numerous attempts have been made to replace animal serum as a cell culture medium supplement. Some of these attempts were made from human plasma fractions, particularly fractions using the Cohn method. Despite which, at present animal serum continues to be widely used as a supplement for mammalian cell culture media and attempts to replace it with a derivative of human plasma have failed.
From all of the above, it can be deduced that in the prior art no material of human origin is available which could be used to supplement cell culture media and which is also safe as regards the transmission of pathogenic agents, can be obtained on an industrial scale at an acceptable cost and profit, has adequate uniformity between batches and is of pharmaceutical grade quality.